一般口演

一般口演睡眠と生体リズム Sleep and Biological Rhythms 座長：山中章弘（名古屋大学環境医学研究所）

2022年6月30日　9:00～9:15　沖縄コンベンションセンター会議場A2　第7会場 1O07m1-01 恒常的睡眠要求を制御する神経細胞内新規シグナル伝達経路 Novel signaling pathways regulating homeostatic sleep need in neurons 北園智弘(1)、堀田（平島）範子(1)、岩崎加奈子(1)、藤山知之(1)、船戸弘正(1,2)、柳沢正史(1,3,4) 1. 筑波大学、2. 東邦大・医・解剖微細形態、3. テキサス大・サウスウェスタン医学センター、4. 筑波大・TARA Tomohiro Kitazono(1), Noriko Hotta-Hirashima(1), Kanako Iwasaki(1), Tomoyuki Fujiyama(1), Hiromasa Funato(1,2), Masashi Yanagisawa(1,3,4) 1. Univ of Tsukuba, 2. Dept of Anat, Fac of Med, Toho Univ, 3. Dept of Mol Genet, Univ of Texas Southwestern Med Cent, Dallas, USA, 4. TARA, Univ of Tsukuba Keyword: sleep, signaling pathway, kinase, GTPase Sleep is a behavior ubiquitously conserved from vertebrates to invertebrates. In sleep/wake regulation, homeostatic sleep need accumulates during wakefulness and dissipates during sleep. Despite the importance, the molecular/cellular basis of homeostatic sleep need remains largely unclear. We recently reported that: first, salt-inducible kinase 3 (SIK3) signaling in neurons regulates inherent sleep need (Funato et al., Nature, 2016); second, cumulative phosphorylation states of a specific subset of mostly synaptic proteins are correlated with changes in sleep need (Wang et al., Nature, 2018). Taken together, we propose that changes of sleep need can be translated into cumulative changes of phosphorylation states of certain brain proteins through SIK3 signaling pathways in neurons. In this study, we aim to reveal the molecular/cellular basis of homeostatic sleep need by identifying upstream and downstream components of SIK3 in sleep/wake regulation. As a possible upstream regulator of SIK3, we focused on liver kinase B1 (LKB1). The activation of SIK3 kinase activity requires phosphorylation of the kinase domain at its T-loop residue (T221), and LKB1 can phosphorylate this residue. To investigate whether LKB1 functions upstream of SIK3 in sleep/wake regulation, we generated postnatal neuron-specific LKB1 knockout (nKO) mice. LKB1 nKO caused profound decrease in total NREM sleep (NREMS) time and EEG delta power during NREMS, an index of sleep need. This phenotype is opposite from that seen in the SIK3 gain-of-function (Sleepy) mutant, and similar to SIK3 loss-of-function mutants in flies, worms, and mice. These results suggested that SIK3 kinase activity is correlated to inherent sleep need. Furthermore, when constitutively active mutation of SIK3 (T221E) was induced to LKB1 nKO mice, weaker EEG delta power during NREMS was rescued. These results confirm that LKB1 regulates sleep/wake behavior by acting upstream of SIK3. Downstream components of SIK3 have not been well investigated. As the first step of identifying the downstream signaling pathway of SIK3, we conducted in vitro substrate screening of SIK3 by kinase-oriented substrate screening (KIOSS) method (Nishioka et al., 2015) and pathway analysis. These analyses showed that the Rho-GTPase signaling pathway is strongly related to phosphoproteins identified in the screening. Our studies provide novel insight into sleep/wake regulation.		2022年6月30日　9:15～9:30　沖縄コンベンションセンター会議場A2　第7会場 1O07m1-02 SIK3の機能獲得型変異が睡眠量を増加させる神経細胞集団の同定 Neuroanatomical analysis of how the gain-of-function mutation in Sik3 increases sleep 岩﨑加奈子(1)、藤山知之(1)、中田慎也(1)、パクミンジョン(1)、三好千香(1)、堀田-平島範子(1)、一久綾(1)、柿﨑美代(1)、石川有紀子(1)、杉山文博(2)、水野聖哉(2)、阿部学(3)、﨑村建司(3)、高橋智(2)、船戸弘正(1,4)、柳沢正史(1,5,6) 1. 筑波大学　国際統合睡眠医科学研究機構、2. 筑波大学　生命科学動物資源センター、3. 新潟大学脳研究所　生命科学リソース研究センター、4. 東邦大学　医学部　解剖学講座　微細形態学分野、5. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Texas, USA、6. 筑波大学　生存ダイナミクス研究センター Kanako Iwasaki(1), Tomoyuki Fujiyama(1), Shinya Nakata(1), Minjeong Park(1), Miyoshi Chika(1), Noriko Hotta-Hirashima(1), Aya Ikkyu(1), Miyo Kakizaki(1), Yukiko Ishikawa(1), Fumihiro Sugiyama(2), Seiya Mizuno(2), Manabu Abe(3), Kenji Sakimura(3), Satoru Takahashi(2), Hiromasa Funato(1,4), Masashi Yanagisawa(1,5,6) 1. IIIS, Univ of Tsukuba, Ibaraki, Japan, 2. LARC, Univ of Tsukuba, Ibaraki, Japan, 3. Department of Animal Model Development, BRI, Niigata University, Niigata, Japan, 4. Department of Anatomy, Faculty of Medicine, Toho University, Tokyo, Japan, 5. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Texas, USA, 6. Life Science Center for Survival Dynamics, TARA, Univ of Tsukuba, Ibaraki, Japan Keyword: SLEEP, KINASE, SIK3, HYPOTHALAMUS Sleep is evolutionary conserved behavior across animal kingdom. Sleep amount is tightly regulated in a homeostatic manner, but the mechanism is largely unknown. Recently, we found a gain-of-function Sleepy (Slp) mutation in the Sik3 gene, which produces the mutant SIK3(SLP) protein, increases sleep amount and non-REM sleep (NREMS) EEG delta density, an index of sleep need. SIK3 is expressed and required for various physiological functions in multiple tissues and organs. Since sleep is affected by peripheral organs and tissues, it remains to be elucidated whether neurons or other types of cells are responsible for the increased NREMS in Sleepy mutant mice. Here, we investigated whether SIK3(SLP) in neurons are sufficient to produce sleep phenotype of sleepy mutant mice. We newly developed Synapsin1^CreERT2;Sik3^Sleepy-floxmice to induce Sik3(Slp) in neurons upon tamoxifen injections. Histological examinations showed that CreERT2 expression recapitulate endogenous Synapsin1 pattern, and tamoxifen administration induce neuron specific recombination. Synapsin1^CreERT2;Sik3^Sleepy-floxmice administrated tamoxifen at late infancy exhibited increased NREMS time and NREMS EEG delta density. In addition, simulated sleep-homeostatic Process S suggested that sleep need of Sik3^Slp and Synapsin1^CreERT2;Sik3^Sleepy-floxmice tend to remain high level. These results suggested that SIK3 plays important role regulating sleep amounts and sleep need in mature neurons. Furthermore, we explored neural populations which increased NREMS amounts upon SIK3(SLP) expression with AAV vectors. We found that SIK3(SLP) expression in hypothalamus increased NREMS amounts, but not NREMS EEG delta density. These results imply that neural populations that increase NREMS and NREMS EEG delta power in Sleepy mutant mice are not identical. Now, we are investigating whether SIK3(SLP) in the hypothalamus is necessary for increasing NREMS, and markers of the responsive brain region.

2022年6月30日　9:30～9:45　沖縄コンベンションセンター会議場A2　第7会場 1O07m1-03 SIK3キナーゼ活性を介した睡眠覚醒制御機構の解明 Molecular mechanisms for sleep/wake regulation mediated by SIK3 kinase activity 中田慎也(1)、阿部-小宮春奈(1)、藤山知之(1)、三好千香(1)、一久綾(1)、浅野冬樹(1)、水野聖哉(2)、杉山文博(2)、高橋智(2)、船戸弘正(1,3)、柳沢正史(1) 1. 筑波大学国際統合睡眠医科学研究機構、2. 筑波大学医学医療系、3. 東邦大学医学部解剖学講座 Shinya Nakata(1), Haruna Abe-Komiya(1), Tomoyuki Fujiyama(1), Chika Miyoshi(1), Aya Ikkyu(1), Fuyuki Asano(1), Seiya Mizuno(2), Fumihiro Sugiyama(2), Satoru Takahashi(2), Hiromasa Funato(1,3), Masashi Yanagisawa(1) 1. International Institute for Integrative Sleep Medicine, University of Tsukuba, 2. Faculty of Medicine, University of Tsukuba, 3. Department of Anatomy, Toho University Keyword: sleep, kinase, SIK3 Sleep is a well-conserved behavior regulated by sleep need in a homeostatic manner. Sik3 was recently identified as a gene regulating sleep/wakefulness through forward-genetics approach. The pedigree, Sleepy, which expresses SIK3 lacking exon13-encoded region (Δex13), exhibits increased sleep need and prolonged sleep time. On the other hand, SIK3 kinase activity is tightly regulated by phosphorylation of T221 in the kinase domain. The phosphorylation of T221 is increased in wild-type mice after sleep deprivation, suggesting that SIK3 kinase activity increases in mice with a higher sleep need. However, how SIK3 kinase activity is involved in sleep regulation remains unknown. 　Here, we examined the role of SIK3 kinase activity in sleep/wake regulation using mice carrying the T221A or T221E mutation in Sik3 allele. First, to investigate the kinase activity of SIK3 mutants carrying T221A or T221E, we performed in vitro kinase assay using recombinant SIK3 expressed in HEK293T cells. While SIK3 carrying T221A showed almost no kinase activity, SIK3 carrying T221E showed decreased but significant kinase activity. Next, we performed EEG/EMG-based sleep/wake analysis for SIK3(T221A) and SIK3(T221E) mice. SIK3(T221A) heterozygous mice showed decreased EEG delta density during NREM sleep, an indicator of sleep need. On the other hand, SIK3(T221E) homozygous mice also tended to show decreased NREM sleep delta density, consistent with the kinase activity. Those mutant mice did not show any changes in total sleep time. These results suggest that SIK3 kinase activity is required for the regulation of sleep need. Further, to examine the role of SIK3 kinase activity in Sleepy mutant mice, we analyzed sleep/wake behavior of SIK3(T221A-Δex13) mice and SIK3(T221E-Δex13) mice. SIK3(T221A-Δex13) heterozygous mice showed decreased NREM sleep delta density and sleep time compared with SIK3(Δex13) heterozygous mice. On the other hand, SIK3(T221E-Δex13) homozygous mice showed increased sleep time compared with SIK3(T221E) homozygous mice. These results suggest that, while SIK3 kinase activity is required for increased sleep need and amount, Sleepy mutation induces another functional alternation for increased sleep time.		2022年6月30日　9:45～10:00　沖縄コンベンションセンター会議場A2　第7会場 1O07m1-04 視交叉上核の神経ネットワークにより駆動されるcAMPの概日リズム Network driven circadian cAMP rhythm in the mouse suprachiasmatic nucleus 小野大輔(1)、Hung Chi Jung(1)、山中章弘(1)、杉山崇(2) 1. 名古屋大学、2. オリンパス株式会社 Daisuke Ono(1), Chi Jung Hung(1), Akihiro Yamanaka(1), Takashi Sugiyama(2) 1. Nagoya University, 2. Olympus Corporation Keyword: Circadian rhythm, Suprachiasmatic nucleus, Neural network, cAMP The circadian clock coordinates the temporal timing of physiology and behavior. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus plays a crucial role in the regulation of the temporal timing of physiological functions such as sleep and wakefulness. Individual SCN neurons show intrinsic circadian rhythms that are considered to be regulated by transcription-translation negative feedback loops involving clock genes. Second messengers such as cAMP and Ca²⁺ play a variety of roles in biological functions. Signaling mediates signals from receptors on the cell surface to target molecules inside the cell and amplifies the signaling. Then, the signal alters gene expression and finally changes cellular functions. In the mammalian SCN, several neurotransmitters are suggested to be involved in the SCN neuronal network. Receptors of these ligands are coupled with G-protein and second messenger signaling, such as cAMP and Ca²⁺. In the SCN, intracellular cAMP and Ca²⁺ are considered to be involved in the input and/or output from molecular circadian clock and/or in the circadian oscillations in the SCN using a pharmacological approach. However, the functional roles of cAMP and Ca²⁺ and its dynamics in the SCN neuronal network remain largely unclear. In the present study, we simultaneously visualized the circadian rhythms of intracellular cAMP and Ca²⁺ in the SCN using bioluminescence (Okiluc-aCT) and fluorescence (GCaMP6s), and found that cAMP but not Ca²⁺ rhythms were driven by neuronal networks. We also found that optical manipulation of intracellular cAMP levels in the SCN shifted molecular circadian rhythms. Together, our study demonstrates that intracellular cAMP is a key molecule in the composition of the SCN circadian neuronal network.